Osteoporosis affects 10 million Americans and another 34 million are osteopenic and at risk for developing osteoporosis. Bone fractures, the most important complication of the disease, cause substantial morbidity and mortality in the aging population as well as significant socio-economic cost. Since the 1960's, bisphosphonate drug therapy has produced clinical benefits such as improved bone density and reduced fracture risk by slowing osteoclastic bone resorption. The existing anti-resorptive drugs are plagued with untoward side effects and limited duration of clinical benefits. New and improved strategies for therapeutic intervention in osteoporosis are needed, particularly in the area of new medicines that safely promote anabolic bone growth. Presently, there is only one FDA approved bone anabolic agent, Forteo (teriparatide) that confers significant clinical benefits in osteoporosis, but its use is severely restricted due to safety concerns. Multipotent mesenchymal stem cells (MSCs) are precursors of a variety of cell types, including osteoblasts and adipocytes. Formation of new bone is driven by osteoblastic differentiation of MSCs, a process that can be disrupted by age and other factors in favor of adipogenesis. Parhami et al. discovered that specific naturally- occurring oxysterols induce osteogenesis when applied to MSCs while inhibiting their adipogenesis. Recently, we have characterized a new series of semi-synthetic analogues of the natural oxysterols with improved properties. Our most advanced compound, OXY133, displays increased potency for osteogenic differentiation in vitro, including in mouse and human primary MSCs, and it stimulates robust localized bone formation in vivo in a rat spine fusion model. Here we propose to begin evaluating Oxy133 as a bone anabolic agent in the context of systemic administration and bone targeting. Our strategy involves conjugation of OXY133 to a known bone-targeting agent (BTA) derived from tetracycline. We predict that systemic dosing of such conjugates will result in their selective deposition in bone followed by enzymatic linker hydrolysis and release of the osteogenic agent, OXY133, at controlled rates into the bone tissue. We have produced the first example of such a conjugate, OXY149, and determined that it retains significant osteogenic activity in C3H10T1/2 cells. Expanding on this proof of principle study, we propose to conjugate the BTA-linker to OXY133 through tunable succinate and aspartate ester linkages in different positions of the molecule, likely to result in variable degrees of linker hydrolysis and bone affinity thus allowing for optimization of these properties. We propose to perform studies as part of three Specific Aims: 1) Synthesis of Oxy133-BTA conjugated analogues, 2) Examination of the hydroxyapatite binding capacity of Oxy133-BTA conjugated analogues, and 3) Examination of the osteogenic activity of Oxy133-BTA conjugated analogues. Information obtained from these studies will provide the rationale for future investigation of the therapeutic effects of Oxy133-BTA analogues in animal models of osteoporosis that may ultimately lead to the development of new bone anabolic agents fit for use in humans.